Ultrasonic-assisted extraction, structure and antitumor activity of polysaccharide from Polygonum multiflorum

Ultrasonic-assisted extraction, structure and antitumor activity of polysaccharide from Polygonum multiflorum

Accepted Manuscript Title: Ultrasonic-assisted extraction, structure and antitumor activity of polysaccharide from Polygonum multiflorum Author: Weili ...

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Accepted Manuscript Title: Ultrasonic-assisted extraction, structure and antitumor activity of polysaccharide from Polygonum multiflorum Author: Weili Zhu Xiaoping Xue Zhanjun Zhang PII: DOI: Reference:

S0141-8130(16)30472-X http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.05.061 BIOMAC 6122

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

26-1-2016 14-3-2016 15-5-2016

Please cite this article as: Weili Zhu, Xiaoping Xue, Zhanjun Zhang, Ultrasonicassisted extraction, structure and antitumor activity of polysaccharide from Polygonum multiflorum, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.05.061 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ultrasonic-assisted extraction, structure and antitumor activity of polysaccharide from Polygonum multiflorum Weili Zhu a, Xiaoping Xue a*, Zhanjun Zhang b a

Department of Blood Transfusion, Subei People's Hospital of Jiangsu Province,

Yangzhou 225001, Jiangsu, China b

College of Biological and Chemical Engineering, Yangzhou Vocational University,

Yangzhou 225009, Jiangsu, China 

Corresponding author. Tel: +86-514-87373646, E-mail: [email protected]

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Highlights Ultrasonic-assisted extraction condition was optimized by RSM. Maximum extraction yield of 5.49% was obtained under the optimal extraction condition. A purified polysaccharide with molecular weight of 3.26 × 105 Da was obtained. PPS was characterized as (1→6)--D-glucan by instrumental analysis. PPS exhibited antitumor activity in vitro on HepG-2 and BGC-823 cells.

Abstract Polygonum multiflorum is a popular Chinese herbal medicine with various pharmacological functions. In this study, the ultrasonic-assisted extraction condition, structural characterization and antitumor activity of a polysaccharide from roots of P. multiflorum were investigated. The ultrasonic-assisted extraction condition was optimized by single-factor experiments and response surface methodology. Results showed that the maximum extraction yield (5.49%) was obtained at ultrasonic power 158 W, extraction temperature 62 °C, extraction time 80 min and ratio of water to material 20 mL/g. The obtained crude polysaccharides were further purified to afford a neutral and an acidic fraction. The structure of the main neutral polysaccharide (named PPS with molecular weight of 3.26 × 105 Da) was characterized as a linear (1 →6)--D-glucan by gas chromatography, Fourier transform-infrared spectroscopy, methylation analysis, 1D and 2D nuclear magnetic resonance. At the concentration of 400 g/mL, the inhibitory ratios of PPS on HepG-2 and BGC-823 cells were 53.35%

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and 38.58%, respectively. Results suggested this polysaccharide could be a potential natural antitumor agent. Keywords: Polysaccharides; Polygonum multiflorum; Ultrasound-assisted extraction; Structural characterization

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1. Introduction Heshouwu, the dried root of Polygonum multiflorum Thunb., is one of the most popular Chinese herbal medicines because of its pharmacological functions [1]. In the Chinese Pharmacopoeia, there are two forms of Heshouwu decoction pieces: the raw state of root (P. multiflorum) and processed form of root (Polygoni multiflori Radix Praeparata) [2]. Till now, many pharmacological studies and clinical practices have demonstrated that Heshouwu possess various biological activities, including antiaging, immunomodulating, antihyperlipidaemia, hepatoprotective, antiinflammatory and anticancer effects etc. [3–4]. These valuable pharmacological functions should be attributed to a variety of constituents, such as stilbene glycosides, anthraquinones, phenolics, flavonoids, phospholipids and carbohydrate compounds [3–5]. Recently, polysaccharides have been domenstrated as one kind of the main bioactive components in Heshouwu [6–8]. Chen reported that intraperitoneal administration of water soluble polysaccharides from P. multiflori could increase levels of serum IL-2 and hematological parameters, enhance antioxidant profiles, and promote hematopoiesis of splenocytes [6]. Lv revealed that polysaccharides from P. multiflorum possessed strong antioxidant capacity against free radical, lipid oxidation and protein glycation [7]. Zhang suggested the alkali-extractable polysaccharide from P. multiflori exhibited significant immunomodulation activity by activating splenocytes and peritoneal macrophages, and also protecting these immunocytes against immunosuppression induced by 5-Fu [8]. However, little attention has been paid to the extraction condition, structural characterization and other bioactive

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properties of polysaccharides from Heshouwu. Polysaccharides are usually extracted from plant materials by hot water extraction method, which requires long extraction time and high extraction temperature [9]. Compared with the conventional hot water extraction method, ultrasonic-assisted extraction is one of the most promising techniques with many advantages, such as shorter extraction time, less solvent used and higher extraction rate [10]. Till now, ultrasonic-assisted extraction has been widely used to extract polysaccharides from different plant materials. Moreover, response surface methodology (RSM) has been extensively applied to optimize extraction conditions of polysaccharides [11–14]. The main advantage of RSM is the reduced number of experimental trials needed to evaluate multiple parameters and their interactions. In this study, ultrasonic-assisted extraction method was used to extract polysaccharides from the roots of P. multiflorum. The extraction condition of crude polysaccharides was optimized by RSM. The obtained crude polysaccharides were further purified and characterized by many instrumental methods, including gas chromatography (GC), Fourier transform-infrared (FT-IR), methylation analysis and nuclear magnetic resonance (NMR). The inhibitory effect of polysaccharide against tumor cell proliferation was also determined. This study provides novel information on extraction condition, structural characterization and antitumor activity of polysaccharides from P. multiflorum. 2. Materials and Methods 2.1. Materials and reagents

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The dried roots of P. multiflorum were purchased from Xinyang Medicine Company (Henan, China). Dextrans T-series standards (T-500, T-200, T-100, T-50 and T-10) were purchased from Pharmacia Co. Ltd. (Uppsala, Sweden). Deuterium oxide (D2O), penicillin and streptomycin were all purchased from Sigma Chemical Co. (MO, USA). Human hepatoma HepG-2 cells and gastric cancer BGC-823 cells were obtained from the Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China). Fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco BRL (NY, USA). All other reagents were of analytical grade. 2.2. Ultrasound-assisted extraction (UAE) of polysaccharides from P. multiflorum The dried roots of P. multiflorum were ground in a blender to obtain a fine powder, defatted and decolored twice with 80% ethanol at 60 ◦C for 2 h in a Soxhlet extractor system, and then centrifuged at 10, 000 rpm for 15 min. The precipitate was collected, dried and used for the extraction of polysaccharides in an ultrasonic cleaner (SB-5200DTD, Xinzhi Biotech Co., Ningbo, China). Each dried pretreated sample (20 g) was extracted in a designed ultrasonic power (80–160 W), extraction temperature (30–70 ◦C), extraction time (20–100 min) and water-to-raw material ratio (5–25 mL/g) by single-factor experimental design. The sonicated mixture was centrifuged at 10, 000 rpm for 15 min, and the supernatant was collected, deproteinized by Sevag method for 5 times, dialyzed against distilled water for 72 h and freeze-dried to obtain crude polysaccharide [15]. The extraction yield of polysaccharides was calculated by the following formula:

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Extraction yield (%) = (W1/W0) × 100

(1)

where W1 and W0 are the weights of crude polysaccharides and dried powder of P. multiflorum roots, respectively. 2.3. Optimization of extraction condition by Box-Behnken design (BBD) On the basis of the results of single-factor experiments, a 29-run BBD with four variables (ultrasonic power, extraction temperature, extraction time and ratio of water to material) and three levels including five replicates at the centre point was used to optimize the extraction condition. As shown in Table 1, four variables were designated as A, B, C and D, and three levels were coded as +1, 0, and −1 for high, intermediate and low levels, respectively. Design-Expert software (version 8.0, Stat-Ease Inc., Minneapolis, USA) was adopted to analyze the experimental data. The relationships and interrelationships of the variables were based on the following second-order polynomial model: Y = β0 + ∑ βixi + ∑ βiixi2 + ∑ βijxixj

(2)

where Y was the predicted response (extraction yield of crude polysaccharides), xi and xj were the level of independent variable, β0, βi, βii, and βij were the model intercept, linear, quadratic and interaction coefficients, respectively. 2.4. Purification of crude polysaccharides Crude polysaccharide solution (10 mg/mL, 5 mL) was firstly purified on DEAE-52 column (2.6 × 30 cm). The column was stepwise eluted by distilled water, 0.1, 0.3, 0.5 and 1 M NaCl solutions at a flow rate of 60 mL/h. Elute (10 mL/tube) was collected automatically and the carbohydrates were determined by the 28

phenol-sulfuric acid method using glucose as the standard [15]. The main polysaccharide fraction (named F-1) was pooled, concentrated, dialyzed, freeze-dried and further purified on Sepharose CL-4B column (1.6 × 80 cm). The column was eluted by distilled water at a flow rate of 12 mL/h. The resultant fraction (named PPS) was pooled, concentrated, dialyzed and freeze-dried for further analysis. 2.5. Determination of homogeneity and molecular weight of PPS The homogeneity and molecular weight of PPS was determined by high performance size exclusion chromatography (HPSEC) on Agilent 1100 system (Agilent Technologies, CA, USA) equipped with a TSK gel G4000 PWXL column (30 cm × 7.8 mm × 10 m, Tosoh Corp., Tokyo, Japan) and an evaporative light scattering detector. The column was eluted with distilled water at 50 °C and flow rate of 0.6 mL/min. The column was calibrated with dextran T-series standards (T-500, T-200, T-100, T-50 and T-10) of known molecular weights. Each dextran standard solution was prepared by dissolving 10 mg of sample in 5 mL distilled water. PPS solution was prepared in the same fashion. The dextran standard solutions were then separately injected into the column and a linear regression standard curve of log molecular weight versus HPSEC retention time was calculated. The linear regression equation of standard curve was as follows: Lg(Mw) = −1.14Tr + 15.89

(3)

where Mw represents the molecular weight, and Tr represents retention time. Afterwards, the PPS solution was injected into the column and the molecular weight of PPS was determined according to the above standard curve.

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2.6. Determination of protein, uronic acid, sulfuric group and polyphenols contents in PPS The protein content in polysaccharide was determined by the method of Bradford using bovine serum albumin as the standard [17]. The uronic acid content was measured by m-hydroxydiphenyl colorimetric method using D-glucuronic acid as the standard [18]. The sulfate group content was determined by the method of barium chloride–gelatin

[19]. The total

polyphenols

content was

determined

by

Folin-Ciocalteu method using gallic acid as the standard [20]. 2.7. Structural characterization of PPS 2.7.1. Monosaccharide composition analysis The monosaccharide composition of PPS was determined according to the reported method [21]. PPS (6 mg) was hydrolyzed in 2 mL of 2 M trifluoroacetic acid at 120 °C for 2 h. The hydrolyzate was concentrated to dryness and then converted to its trimethylsilyl derivative by adding 1 mL of pyridine, 0.4 mL of hexamethyldisilazane and 0.2 mL of trimethylchlorosilane and reacting at 80 °C for 30 min. The trimethylsilyl derivative of PPS hydrolyzate was further analyzed on Agilent 6890A GC system (Agilent Technologies, CA, USA) equipped with flame ionization

detector

and

a

HP-5

fused-silica

capillary

column

(30 m × 0.25 mm × 0.25 μm). The following chromatographic conditions were applied: initial column temperature was held at 100 °C for 5 min, then programmed at a rate of 5 °C/min to 150 °C and held at 150 °C for 5 min, subsequently programmed at 5 °C/min to 240 °C and held at 240 °C for 2 min. The temperature of injector and

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detector were set at 250 °C and 280 °C, respectively. Nitrogen gas was used as the carrier gas with a flow rate of 1 mL/min. The trimethylsilyl derivatives of monosaccharide standards with erythritol as the internal standard were also derivated and subjected to GC analysis in the same way. 2.7.2. FT-IR The FT-IR spectroscopy of PPS was analyzed on a continuous scan Tensor-27 spectrometer (Bruker Optics, Wissembourg, France) in the 4000–400 cm−1 region via the KBr pressed disk method. 2.7.3. Methylation and GC-MS analysis Methylation of PPS was performed according to the method of Ciucanu and Kerek [22]. Complete methylation was confirmed by the lack of hydroxyl peak in FT-IR spectrum. The permethylated polysaccharide was hydrolysed with trifluoroacetic acid, reduced with NaBH4 and acetylated with acetic anhydride. The resulting partially methylated alditol acetate derivatives were analyzed on a Varian CP-3800 gas chromatograph coupled with a Saturn 2000 ion trap mass spectrometer (Walnut Creek, CA, USA). A DB-5 fused silica capillary column (30 m × 0.25 mm × 0.25 mm, J&W Scientific, Folsom, CA, USA) was used with helium as the carrier gas. 2.7.4. NMR The freeze-dried PPS was dissolved in D2O and subjected to NMR analysis on an AVANCE-600 spectrometer (Bruker Inc., Ettlingen, Germany) at 25 °C. The operating frequencies were 600 MHz for 1H NMR and 150 MHz for

13

C NMR, respectively.

The 2D NMR spectra of 1H/1H homonuclear correlation spectroscopy (COSY) and

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heteronuclear single quantum coherence (HSQC) were also recorded. 2.8. Antitumor activity of PPS The antitumor activity of PPS on human hepatoma HepG-2 cells and gastric cancer BGC-823 cells was determined by the MTT-based colorimetric method [23]. HepG-2 and BGC-823 cells were firstly cultured in DMEM medium supplemented with 10% FBS, 100 U/mL of penicillin and 100 mg/L of streptomycin under humidified atmosphere containing 5% CO2 at 37 °C. Then, 100 L of tumor cells with a density of 2 × 105 cells/mL were pipetted into a 96-well plate. After inoculation under 5% CO2 at 37 °C for 24 h, the tumor cells were treated with various concentrations of PPS (50, 100, 200 and 400 g/ml in fresh medium, 50 L/well) for 48 h. Subsequently, 10 L of MTT (5 mg/mL) was added into each well and the plate was incubated for additional 4 h. Finally, the liquid was removed and 100 L of DMSO was added into each well to dissolve the formed crystal formazan. The absorbance was measured by a Synergy 2 multi-mode microplate reader (BioTek Instruments, Inc., VT, USA) at 570 nm. 5-Fluorouracil (5-FU, 50 g/mL) was used as the positive control. The inhibitory ratio of PPS against tumor cell proliferation was calculated by the following formula: Inhibitory ratio (%) = [1 – (Asample – Ablank)/(Acontrol – Ablank)] × 100

(4)

where Acontrol and Ablank were the absorbance of the system without addition of PPS and tumor cells, respectively. 2.9. Statistical analysis Data were expressed as mean ± standard deviation (SD) of triplicates. Statistical

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analysis was performed using Design-Expert software of version 8.0 (Stat-Ease Inc., Minneapolis, USA) and SPSS software of version 13.0 (Chicago, IL, USA). Difference was considered to be statistically significant if P < 0.05. 3. Results and discussion 3.1. Effects of extraction parameters on the extraction yield of crude polysaccharides Effects of four different extraction parameters (ultrasonic power, extraction temperature, extraction time and ratio of water to material) on the extraction yield of crude polysaccharides were shown in Fig. 1. The extraction yield increased from 2.33% to 4.21% as ultrasonic power increased from 80 to 140 W (Fig. 1a). This might be due to the increase of the mass transfer rate of polysaccharides with high ultrasonic power [11]. However, no significant change was observed in the extraction yield as ultrasonic power continued to increase. As shown in Fig. 1b, the extraction yield increased rapidly from 2.90% to 4.72% as extraction temperature increased from 30 to 60 °C, indicating high temperature could facilitate polysaccharides releasing from cells to the exterior solvent. When extraction time increased from 20 to 60 min, the extraction yield significantly increased to 4.77% and slightly decreased as extraction time was over 80 min (Fig. 1c). Moreover, Fig. 1d showed the maximum extraction yield was achieved at the ratio of water to material 20 mL/g. Based on the above results, ultrasonic power 140 W, extraction temperature 60 °C, extraction time 60 min and ratio of water to material 20 mL/g were chosen as the centre points for the following BBD. Fig. 1

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3.2. Optimization of extraction condition by RSM RSM was used to optimize the extraction condition of polysaccharides from P. multiflorum. The experimental design and the corresponding results of BBD were shown in Table 1. Based on the multiple regression analysis, the predicted response variable could be obtained by the following second-order polynomial equation: Y = 5.26 + 0.16A + 0.16B + 0.27C + 0.18D + 0.14AB + 0.43AC −0.04AD − 0.03BC + 0.27BD + 0.23CD − 0.35A2 − 0.72B2 − 0.28C2 − 0.22D2

(5)

where Y was the extraction yield of polysaccharides, A, B, C and D were the coded variables for the ultrasonic power, extraction temperature, extraction time and ratio of water to material, respectively. Table 1 The statistical significance of the regression model was checked by F-test and the analysis of variance (ANOVA) for the response surface quadratic model was shown in Table 2. F-test showed the model had a high F-value (11.83) and a low P-value (< 0.0001), indicating that the model was highly significant. The value of determination coefficient R2 was 0.9221, indicating 92.21% of the variations could be explained by the model. Moreover, a low value of coefficient of the variation (4.46%) indicated high degree of precision and good deal of reliability for the experimental values. The P-value was used to check the significance of each coefficient and the strength of interaction between variables. When P-value was less than 0.05, the model terms were significant. Accordingly, A, B, C, D, AC, BD, CD, A2, B2, C2 and D2 were all significant model terms.

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Table 2 The predicted models were presented in Fig. 2 as 3D response surface plots and Fig. 3 as contour plots. In general, the response surface with circular contour plot indicates the interaction between the corresponding variables is negligible, whereas elliptical or saddle nature of the contour plots indicates the interactions between the corresponding variables is significant [24]. In this case, the interaction terms of AC, BD and CD were significant. The optimum levels of four variables were obtained by analyzing the response surface contour plots using Design-Expert software. The model predicted the maximum extraction yield (5.52%) could be obtained under the following optimum extraction condition: ultrasonic power 158 W, extraction temperature 62 °C, extraction time 80 min and ratio of water to material 20 mL/g. To validate the adequacy of model equation, the verification experiment was carried out under the predicted optimum extraction condition. The actual mean value of extraction yield was 5.49%, which was in good agreement with the predicted value. These results indicated the suitability of the model employed and the success of RSM for optimizing the extraction condition. Fig. 2 Fig. 3 3.3. Purification of crude polysaccharides The crude polysaccharides were firstly purified on a DEAE-52 anion-exchange chromatography to afford a neutral and an acidic polysaccharide, named as F-1 and F-2, respectively (Fig. 4a). The main fraction (F-1) was collected and further purified

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on a Sepharose CL-4B gel filtration chromatography to generate only one single elution peak, named as PPS (Fig. 4b). The recovery ratio of PPS was 70.8% based on crude polysaccharides. The homogeneity and molecular weight of PPS was determined by HPSEC. As shown in Fig. 5, PPS presented only one symmetrical peak on HPSEC, indicating that no other polysaccharide was in the sample. According to the calibration curve of the elution times of dextran T-series standards, the molecular weight of PPS was measured as 3.26 × 105 Da. Lv et al. obtained two polysaccharides with molecular weights of 4.8 × 105 and 6.1 × 105 Da from P. multiflorum by conventional hot water extraction method [7]. By contrast, the molecular weight of PPS obtained by ultrasonic-assisted extraction was relatively lower than that of polysaccharides obtained by hot water extraction, which was probably due to the degradation effect of ultrasound treatment. In addition, PPS showed a negative iodine–potassium iodide reaction, indicating that it was not a starch-type polysaccharide. Further chemical analysis showed that PPS did not contain any protein, uronic acid, sulfuric groups and polyphenols. Fig. 4 Fig. 5 3.4. Structural characterization of PPS In the past, most investigators merely focused on the monosaccharide composition of polysaccharides from Heshouwu [6–8]. However, the detailed structure features of these polysaccharides are still unclear. Therefore, we further characterized the structure of PPS by many instrumental methods.

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Monosaccharide composition of PPS was analyzed by GC. As shown in Fig. 6, PPS was mainly composed of glucose with small amount of other monosaccharides. The molar ratio of arabinose, rhamnose, galactose, glucose and mannose was 1.24: 1.83: 1.03: 30.25: 1.00. Lv and Chen also found that glucose was the main sugar unit for water soluble polysaccharides from P. multiflorum and P. multiflori, respectively [6–7]. Recently, Zhang reported an alkali-extractable polysaccharide from P. multiflori water extracted residue was mainly composed of mannose, rhamnose, galacturonic acid, galactose, glucose, xylose and arabinose with a molar ratio of 0.14: 0.44: 0.15: 4.31: 0.24: 1.06: 0.86 [8]. By comparison, PPS had different monosaccharide compositions and molar ratios with reported polysaccharides from P. multiflorum and P. multiflori, which might be due to the difference in the extraction methods of polysaccharides. Fig. 6 FT-IR spectrum of PPS in KBr pellet was shown in Fig. 7. The strong band at 3390 cm–1 was assigned to the hydroxyl stretching vibration. The band at 2925 cm–1 was due to C–H stretching vibration. The band at 1650 cm–1 was corresponded to associated water. Moreover, bands at 916 and 765 cm–1 are the characteristic of the pyranose form of glucosyl residues [25]. The characteristic absorption at 848 cm–1 indicates the -configuration of glucosyl units [26]. Fig. 7 The fully methylated product of PPS was hydrolyzed, converted into alditol acetates and analyzed by GC–MS. As a result, the major derivative of PPS was

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1,5,6-tri-O-acetyl-2,3,4-tri-O-methylglucitol, indicating (1→6)-linked glucopyranosyl was the main residue of the polysaccharide. 1H and

13

C NMR spectra of PPS were

presented in Fig. 8a. 1H NMR spectrum of PPS exhibited one main anomeric proton at 4.94 ppm, indicating glucosyl residues were α-glycosidically linked, which was in agreement with the result of FT-IR. The -configuration of glucosyl residues could also be confirmed by the presence of anomeric carbon at 97.76 ppm in

13

C NMR

spectrum (Fig. 8b). The chemical shift of H-2 could be assigned from the COSY spectrum (Fig. 9a) according to the principle that H-2 correlates with H-1. In this case, H-3 to H-6 could also be assigned. Therefore, chemical shifts at 3.54, 3.67, 3.47, 3.87, 3.72, and 3.95 ppm can be attributed to H-2, H-3, H-4, H-5, H-6a and H-6b of the glucosyl residues, respectively. The carbon signals of C-2 to C-6 were identified from cross peaks in HSQC spectrum (Fig. 9b) and the literature data [25, 27]. Accordingly, carbon signals at and 65.62 ppm could be assigned to C-3, C-2, C-5, C-4 and C-6 of glucosyl residues, respectively. These results all suggested that the glucosyl residues were linked by -(1→6) glycosidic linkage and PPS was a linear (1→6)--D-glucan. Fig. 8 Fig. 9 3.5. Antitumor activity in vitro of PPS Till now, only few studies have focused on the biological activities of polysaccharides from Heshouwu, such as antioxidant and immunomodulation activities [6–8]. However, the antitumor activity of polysaccharides from P.

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multiflorum and P. multiflori has been rarely reported. In this study, the antitumor activity of PPS on human hepatoma HepG-2 cells and gastric cancer BGC-823 cells was investigated. As shown in Fig. 10, PPS exhibited a concentration dependent inhibition effect on both HepG-2 and BGC-823 cells. At a concentration of 400 g/mL, the inhibitory ratios of PPS against HepG-2 and BGC-823 cells were 53.35% and 38.58%, respectively. By contrast, PPS showed relatively higher inhibitory ratio against HepG-2 than BGC-823 cells. Inhibitory ratios of the positive control (5-FU) at 50 g/mL on HepG-2 and BGC-823 cells were 86.55% and 70.32%, respectively. Our results showed that PPS played an important role on inhibition of tumor cells proliferation. Further investigation on the antitumor mechanism of PPS will be studied in the future. Fig. 10 4. Conclusion In this study, RSM was applied to optimize the ultrasonic-assisted extraction of polysaccharides from the roots of P. multiflorum. Statistical analysis showed ultrasonic power, extraction temperature, extraction time and ratio of water to material were all significant factors affecting the extraction yield of polysaccharides. A neutral polysaccharide with the molecular weight of 3.26 × 105 Da could be sequentially purified by DEAE-52 and Sepharose CL-4B chromatography. This polysaccharide was deduced to be a linear (1→6)--D-glucan according to the results of many instrumental analyses. Moreover, this polysaccharide exhibited inhibition activity on HepG-2 and BGC-823 cell proliferation in vitro. The result suggested this

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polysaccharide could be a potential natural antitumor agent. Acknowledgments This work was supported by the Scientific Research Foundation of Subei People's Hospital of Jiangsu Province.

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References [1] S. Yao, Y. Li, L. Kong, Preparative isolation and purification of chemical constituents from the root of Polygonum multiflorum by high-speed counter-current chromatography, J. Chromatogr. A 1115 (2006) 64–71. [2] M. Wang, R. Zhao, W. Wang, X. Mao, J. Yu, Lipid regulation effects of Polygoni Multiflori Radix, its processed products and its major substances on steatosis human liver cell line L02, J. Ethnopharmacol. 139 (2012) 287–293. [3] L. Lin, B. Ni, H. Lin, M. Zhang, X. Li, X. Yin, C. Qu, J. Ni, Traditional usages, botany, phytochemistry, pharmacology and toxicology of Polygonum multiflorum Thunb.: A review, J. Ethnopharmacol. 159 (2015) 158–183. [4] G.A. Bounda, Y.U. Feng, Review of clinical studies of Polygonum multiflorum Thunb. and its isolated bioactive compounds, Pharmacognosy Res. 7 (2015) 225–236. [5] N. Saewan, A. Jimtaisong, Natural products as photoprotection, J. Cosmet. Dermatol. 14 (2015) 47–63. [6] Q. Chen, S.Z. Zhang, H.Z. Ying, X.Y. Dai, X.X. Li, C.H. Yu, H.C. Ye, Chemical characterization and immunostimulatory effects of a polysaccharide from Polygoni Multiflori Radix Praeparata in cyclophosphamide-induced anemic mice, Carbohydr. Polym. 88 (2012) 1476–1482. [7] L. Lv, Y. Cheng, T. Zheng, X. Li, R. Zhai, Purification, antioxidant activity and antiglycation of polysaccharides from Polygonum multiflorum Thunb, Carbohydr. Polym. 99 (2014) 765–773. [8] Q. Zhang, Y. Xu, S. Zou, X. Zhang, K. Cao, Q. Fan, Novel functional polysaccharides from Radix Polygoni Multiflori water extracted residue: Preliminary characterization and immunomodulatory activity, Carbohydr. Polym. 137 (2016) 625–631. [9] A. Zong, H. Cao, F. Wang, Anticancer polysaccharides from natural resources: A review of recent research, Carbohydr. Polym. 90 (2012) 1395–1410. [10] K. Vilkhu, R. Mawson, L. Simons, D. Bates, Applications and opportunities for ultrasound assisted extraction in the food industry–A review, Innov. Food Sci. Emerg.

41

9 (2008) 161–169. [11] Y. Liu, M. Qiang, Z. Sun, Y. Du, Optimization of ultrasonic extraction of polysaccharides from Hovenia dulcis peduncles and their antioxidant potential, Int. J. Biol. Macromol. 80 (2015) 350–357. [12] K. Afshari, V. Samavati, S.A. Shahidi, Ultrasonic-assisted extraction and in-vitro antioxidant activity of polysaccharide from Hibiscus leaf, Int. J. Biol. Macromol. 74 (2015) 558–567. [13] Z. Wu, H. Li, Y. Yang, H. Tan, Ultrasonic extraction optimization of L. macranthoides polysaccharides and its physicochemical properties, Int. J. Biol. Macromol. 74 (2015) 224–231. [14] A. Ahmad, K.M. Alkharfy, T.A. Wani, M. Raish, Application of Box–Behnken design for ultrasonic-assisted extraction of polysaccharides from Paeonia emodi, Int. J. Biol. Macromol. 72 (2015) 990–997. [15] A.M. Staub, Removal of protein–Sevag method, Methods Carbohydr. Chem. 5 (1965) 5–6. [16] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (1956) 350–356. [17] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [18] N. Blumenkrantz, G. Asboe-Hansen, New method for quantitative determination of uronic acids, Anal. Biochem. 54 (1973) 484–489. [19] H. Ye, K. Wang, C. Zhou, J. Liu, X. Zeng, Purification, antitumor and antioxidant activities in vitro of polysaccharides from the brown seaweed Sargassum pallidum, Food Chem. 111 (2008) 428–432. [20] N. Turkmen, F. Sari, Y.S. Velioglu, Effects of extraction solvents on concentration and antioxidant activity of black and black mate tea polyphenols determined by ferrous tartrate and Folin–Ciocalteu methods, Food Chem. 99 (2006) 835–841. 42

[21] J. Liu, X. Wen, X. Zhang, H. Pu, J. Kan, C. Jin, Extraction, characterization and in vitro antioxidant activity of polysaccharides from black soybean, Int. J. Biol. Macromol. 72 (2015) 1182–1190. [22] I. Ciucanu, F. Kerek, A simple and rapid method for the permethylation of carbohydrates, Carbohydr. Res. 131 (1984) 209–217. [23] K. Wang, W. Li, X. Rui, X. Chen, M. Jiang, M. Dong, Characterization of a novel exopolysaccharide with antitumor activity from Lactobacillus plantarum 70810, Int. J. Biol. Macromol. 63 (2014) 133–139. [24] D. Qiao, B. Hu, D. Gan, Y. Sun, H. Ye, X. Zeng, Extraction optimized by using response surface methodology, purification and preliminary characterization of polysaccharides from Hyriopsis cumingii, Carbohydr. Polym. 76 (2009) 422–429. [25] X. Luo, X. Xu, M. Yu, Z. Yang, L. Zheng, Characterisation and immunostimulatory activity of an -(1→6)-D-glucan from the cultured Armillariella tabescens mycelia, Food Chem. 111 (2008) 357–363. [26] S. Li, D. Wang, W. Tian, X. Wang, J. Zhao, Z. Liu, R. Chen, Characterization and anti-tumor activity of a polysaccharide from Hedysarum polybotrys Hand.-Mazz, Carbohydr. Polym. 73 (2008) 344–350. [27] G. Zhao, J. Kan, Z. Li, Z. Chen, Characterization and immunostimulatory activity of an (1→6)-a-D-glucan from the root of Ipomoea batatas, Int. Immunopharmacol. 5 (2005) 1436−1445.

43

Figure legends Fig. 1. Effects of ultrasonic power (a), extraction temperature (b), extraction time (c) and ratio of water to material (d) on the extraction yield of crude polysaccharides. Different lowercase letters in the same bar chart indicate significant difference (P < 0.05). Each value represents mean ± SD of triplicates. Fig. 2. Response surface plots showing the interactive effects of ultrasonic power (A), extraction temperature (B), extraction time (C) and ratio of water to material (D) on the extraction yield (Y) of crude polysaccharides. Fig. 3. Contour plots showing the interactive effects of ultrasonic power (A), extraction temperature (B), extraction time (C) and ratio of water to material (D) on the extraction yield (Y) of crude polysaccharides. Fig. 4. Stepwise elution profile of crude polysaccharides on DEAE-52 column (a) and

elution profile of polysaccharide fraction (F-1) on Sepharose CL-4B column (b). Fig. 5. HPSEC chromatogram of PPS on TSK gel G4000 PWXL column. Fig. 6 GC chromatograms of trimethylsilyl derivatives of standard monosaccharide mixtrue (a) and PPS hydrolyzate (b). Fig. 7. FT-IR spectrum of PPS in KBr pellet. Fig. 8. 1H NMR (a) and 13C NMR (b) spectra of PPS in D2O. Fig. 9. COSY (a) and HSQC (b) spectra of PPS in D2O. Fig. 10. Inhibitory ratio of PPS against human hepatoma HepG-2 cells and gastric cancer BGC-823 cells. Each value represents mean ± SD of triplicates.

44

(a) 5.00

a

Extraction yield (%)

Extraction yield (%)

4.00

c 3.00

b

b

4.00

a

(b) 5.00 a

d

2.00 1.00

3.00

c d

2.00 1.00

0.00

0.00 80

100

120

140

160

30

Ultrasonic power (W)

50

60

70

(d) 6.00 a

5.00

ab

c

a

ab

20

25

b

bc

5.00

Extraction yield (%)

Extraction yield (%)

40

Extraction temperature (°C)

(c) 6.00

4.00

b

d

3.00 2.00

c 4.00

d

3.00

2.00

1.00 0.00

1.00

20

40

60

80

100

Extraction time (min)

5

10

15

Ratio of water to material (mL/g)

Fig. 1

45

Y (%)

Y (%)

Y (%)

Y (%)

Y (%)

Y (%)

(a) (b)

(c) (d)

(e) (f)

Fig. 2

23

Y (%)

Y (%)

(a)

B (°C)

C (min)

(b)

A (W)

A (W) Y (%)

Y (%)

(d)

D (mL/g)

C (min)

(c)

B (°C) Y (%)

A (W) Y (%)

(e)

D (mL/g)

D (mL/g)

(f)

C (min)

B (°C)

Fig. 3

24

Absorbence (490 nm) 1.60

1.2

NaCl gradient (M) F-1

1.20

1 0.8 0.6

0.80

0.4

0.40

F-2

0.2

0.00

0 0

20

40

60

80

100

40

50

Tubes

(b) Absorbence (490 nm)

1.2

PPS

1 0.8 0.6 0.4 0.2 0 0

10

20

30 Tubes

Fig. 4

25

NaCl gradient (M)

Absorbence (490 nm)

(a)

Signal Voltage (mV)

250 200 150 100 50 0 0

5

10 Time (min)

Fig. 5

26

15

20

200

100

300

D-Glucose

16

500

16

18

18

20

20

22

22

Fig. 6

27

600

(b)

400

24 26 28

24 26 28

D-Mannose

D-Glucose

D-Glactose

D-Xylose

L-Fucose

L-Rhamnose

D-Fructose

1200

D-Mannose

1600

L-Arabinose

Erythitol

2000

D-Glactose

L-Arabinose L-Rhamnose

pA

Erythitol

pA 2400

(a)

800

400

30

30

min

min

80

Transmittance (%)

70 60 50 40

2925

1650

848 916 765 549

1420

30

1020

3390

20 10 0 4000

3600

3200

2800

2400

2000

1600 -1

Wavenumber (cm )

Fig. 7

28

1200

800

400

(a)

6.0

H-1 H-3 H-6a H-2 H-4 H-5 H-6b

4.0

5.0

(b)

3.0

2.0

C-3

C-1

1.0

0.0 (ppm)

C-2 C-5 C-4 C-6

120

110

100

90

80

Fig. 8

29

70

60

50 (ppm)

Fig. 9

30

HepG-2 BGC-823

Inhibitory ratio (%)

80

60

40

20

0 50

100

200

Concentration (g/ml)

Fig. 10

31

400

Table 1 Coded levels and real values (in the parentheses) for the experimental design and results of BBD.

Standard order

A: Ultrasonic power (W)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

–1 (120) +1 (160) –1 (120) +1 (160) 0 (140) 0 (140) 0 (140) 0 (140) –1 (120) +1 (160) –1 (120) +1 (160) 0 (140) 0 (140) 0 (140) 0 (140) –1 (120) +1 (160) –1 (120) +1 (160) 0 (140) 0 (140) 0 (140) 0 (140) 0 (140) 0 (140) 0 (140) 0 (140) 0 (140)

Coded levels (real values) B: C: Extraction Extraction temperature time (min) (°C) –1 (50) 0 (60) –1 (50) 0 (60) +1 (70) 0 (60) +1 (70) 0 (60) 0 (60) –1 (40) 0 (60) +1 (80) 0 (60) –1 (40) 0 (60) +1 (80) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) –1 (50) –1 (40) +1 (70) –1 (40) –1 (50) +1 (80) +1 (70) +1 (80) 0 (60) –1 (40) 0 (60) –1 (40) 0 (60) +1 (80) 0 (60) +1 (80) –1 (50) 0 (60) +1 (70) 0 (60) –1 (50) 0 (60) +1 (70) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60)

32

Extractiom yield (%) D: Ratio of water to material (mL/g) 0 (20) 0 (20) 0 (20) 0 (20) –1 (15) –1 (15) +1 (25) +1 (25) –1 (15) –1 (15) +1 (25) +1 (25) 0 (20) 0 (20) 0 (20) 0 (20) 0 (20) 0 (20) 0 (20) 0 (20) –1 (15) –1 (15) +1 (25) +1 (25) 0 (20) 0 (20) 0 (20) 0 (20) 0 (20)

Observed

Predicted

3.73 4.05 3.93 4.81 4.13 5.12 4.73 4.81 4.13 4.55 5.01 5.28 3.79 4.15 4.53 4.77 4.85 4.02 4.41 5.29 4.43 4.05 4.08 4.77 5.28 5.32 5.18 5.24 5.30

4.01 4.06 4.05 4.65 4.07 5.07 4.90 4.99 4.30 4.70 4.76 5.01 3.80 4.17 4.41 4.66 4.63 4.09 4.31 5.49 4.24 4.02 4.09 4.93 5.26 5.26 5.26 5.26 5.26

Table 2 Analysis of variance (ANOVA) of the regression equation and coefficients. Source

Sum squares

Model A-Ultrasonic power B-Extraction temperature C-Extraction time D-Ratio of water to material AB AC AD BC BD CD A2

7.0129 0.3136

of Degrees of freedom 14 1

Mean square

F-value

P-value (Prob > F)

0.5009 0.3136

11.8322 7.40831

< 0.0001* 0.0165*

0.2914

1

0.2914

6.88333

0.0200*

0.8856

1

0.8856

20.9195

0.0004*

0.4294

1

0.4294

10.1430

0.0066*

0.0784 0.7310 0.0056 0.0036 0.2862 0.2070 0.7961

1 1 1 1 1 1 1

0.0784 0.7310 0.0056 0.0036 0.2862 0.2070 0.7961

1.85188 17.2675 0.13287 0.08504 6.76090 4.89012 18.8048

0.1951 0.0010* 0.7209 0.7749 0.0210* 0.0441* 0.0007*

B2

3.3774

1

3.3774

79.7773

< 0.0001*

C2

0.5189

1

0.5189

12.2565

0.0035*

0.3185 0.5927 0.5804 0.0123 7.6056

1 14 10 4 28

0.3185 0.0423 0.0580 0.0031

7.52282

0.0159*

18.8433

0.0061*

D2 Residual Lack of Fit Pure Error Cor Total

*Statistically significant difference at 0.05 level.

33